Selective activator of human ClpP triggers cell cycle arrest to inhibit lung squamous cell carcinoma

Chemo-activation of mitochondrial ClpP exhibits promising anticancer properties. However, we are currently unaware of any studies using selective and potent ClpP activators in lung squamous cell carcinoma. In this work, we report on such an activator, ZK53, which exhibits therapeutic effects on lung squamous cell carcinoma in vivo. The crystal structure of ZK53/ClpP complex reveals a π-π stacking effect that is essential for ligand binding selectively to the mitochondrial ClpP. ZK53 features on a simple scaffold, which is distinct from the activators with rigid scaffolds, such as acyldepsipeptides and imipridones. ZK53 treatment causes a decrease of the electron transport chain in a ClpP-dependent manner, which results in declined oxidative phosphorylation and ATP production in lung tumor cells. Mechanistically, ZK53 inhibits the adenoviral early region 2 binding factor targets and activates the ataxia-telangiectasia mutated-mediated DNA damage response, eventually triggering cell cycle arrest. Lastly, ZK53 exhibits therapeutic effects on lung squamous cell carcinoma cells in xenograft and autochthonous mouse models.

d Root mean squared deviation.
Supplementary Table 2.The residues with disordered electron density in the X-ray structure of ZK53/HsClpP56 complex.
b The side chains of the residues exhibiting low electron density were truncated.
Supplementary Table 3. Complete blood count analysis of Balb/c mice (n = 4) treated with ZK53.

Primer
Sequence (5'-3') Chloroacetyl chloride (1.13 g, 10 mmol, 1.0 eq) was dissolved in DCM (10 mL) and added dropwise to the reaction mixture.The reaction was monitored by TLC (hexane/EtOAc (5:1)) and stirred at room temperature for 4 h.The mixture was then washed with saturated NaHCO3.The organic layer was separated, and the aqueous layer was washed with DCM.The combined organic solution was dried over anhydrous Na2SO4, the suspension was filtered, and the filtrate was concentrated in vacuo to afford crude compound 2 without further purification.Compound 2, 4-chlorophenol (1.29 g, 10 mmol, 1.0 eq) and K2CO3 (2.07 g, 15 mmol, 1.5 eq) were dissolved in dimethyl sulfoxide (DMSO) and stirred at room temperature overnight, the mixture was washed with water and the organic layer was separated.The combined organic solution was dried over anhydrous Na2SO4, the suspension was filtered and the filtrate was concentrated in vacuo to afford compound 3. Compound 3 was dissolved in 2.0 M hydrochloric acid in ethyl acetate (EA) to 2.0 M and stirred at room temperature for 6 h.The precipitate was filtered and washed with EA to afford compound 4 as a white powder 2.10 g (83 % yield).7, 155.9, 129.5, 126.2, 116.2, 65.5, 42.8, 41.3, 40.3, 38.6.LC-MS [M + H] + 255.1.
Mechanism of selectivity of ZK53 and effects of ZK53 on LrClpP activation and gut microbes' growth.a Presentation of HsClpP monomer with α2 and α4 locating at the periphery and core of HsClpP, respectively.b 2fo-fc map contoured to 1.2σ (blue mesh) showing the electron density of α2 and α4 in our HsClpP structure.The amino acids are presented as yellow sticks.c 2fo-fc density map contoured to 1.0σ (blue mesh) of the ligand-binding area in chain A. d Superimposition of the monomeric structures of HsClpP (blue cartoon, PDB: 1TG6) and ZK53/HsClpP complex (gray cartoon, PDB: 8HGK).e Close view of the ligand-binding pocket in d showing the effect of ZK53 binding on the conformation of key residues.ZK53 is presented as green stick.f Effect of ZK11 and ZK53 on -casein hydrolysis by W146I HsClpP and I91W SaClpP mutants in the PAGE-based assay.The reaction was cultured for 2 h before subjected to PAGE analysis.Representative gels of three independent experiments are shown.g Effect of ADEP 4 on -casein hydrolysis by HsClpP, W146I HsClpP, SaClpP, and I91W SaClpP in the PAGE-based assay.The reaction was cultured for 2 h before subjected to PAGE analysis.Representative gels of three independent experiments are shown.h Effect of ClpP activators on the thermal stability of HsClpP, W146I HsClpP, and I91W SaClpP in the DSF assay (n = 3 biological samples).ΔTm values of ClpP proteins are indicated.i Effect of ONC212 on -casein hydrolysis by HsClpP, W146I HsClpP, SaClpP, and I91W SaClpP in the PAGE-based assay.The reaction was cultured for 2 h before subjected to PAGE analysis.Representative gels of three independent experiments are shown.j The -casein hydrolysis by Y118A ClpP mutant in the PAGE-based assay.Representative gels of three independent experiments are shown.k Effect of ZK53 on -casein hydrolysis by Y118A ClpP in the PAGE-based assay.The reaction was cultured for 2 h before subjected to PAGE analysis.Representative gel of three independent experiments is shown.l Effect of ClpP activators on the thermal stability of HsClpP, and Y118A HsClpP in the NanoDSF assay.ΔTm values of ClpP proteins are indicated (n = 3 biological samples).m Effect of ClpP activators on -casein hydrolysis by LrClpP in the PAGE-based assay.Representative gels of three independent experiments are shown.n Growth curve of gut microbes treated with ClpP activators (n = 3 biological samples).Tetracycline was assayed as a positive control.The data are represented as mean ± SD (error bars).Supplementary Fig. 3 ZK53 inhibits LUSC cell proliferation in a ClpP-dependent manner.a Effect of inducible Y118A ClpP overexpression on colony formation of LUSC cells in clonogenic assay (n = 3 biological samples).The P values are calculated by twosided Student's t test with confidence interval of 95%.b Effect of Y118A ClpP overexpression on proliferation of SK-MES-1, H226, and H1703 cell lines (n = 3 biological samples).The P values are calculated by two-sided Student's t test with confidence interval of 95%.c Quantitative results of clonogenic assay showing the effect of ZK11, ZK53, and ZK24 on colony formation in H1703, H520, SK-MES-1, and H226 cell lines (n = 3 biological samples).d Effect of ZK11 and ZK53 on cell viability of H1703 and H520 cells in growth curve assay (n = 3 biological samples).The P values are calculated by one-way ANOVA with Dunnett's multiple comparisons test.e Representative images showing the inhibitory effect of ZK53 on H520 and SK-MES-1 cells in the EdU proliferation assay (n = 3 biological samples).The percentage of EdU positive (green) cells to total cell count (blue) is calculated.The P values were calculated by a two-sided Student's t-test with confidence interval of 95%.Scale bar, 50 μm.f Inhibitory effect of ZK53 on the cell viability of H1703 and MRC-5 cell lines.The IC50 values are shown (n = 3 biological samples).g Apoptosis analysis of H1703 and MRC-5 cells treated with 1 μM ZK53 for the indicated time points (n = 3 biological samples).The P values were calculated by two-way ANOVA.h The protein level of ClpP in H1703 and SK-MES-1 cell lines with CLPP knockdown.The images shown are representative of three independent experiments.i Effect of ZK11 and ZK53 on the viability of shNS and shCLPP SK-MES-1 cells (n = 3 biological samples).Error bars represent the mean ± SD. j The protein level of ClpP in H1703 and SK-MES-1 cells with CLPP overexpression.The images shown are representative of three independent experiments.k, l Effect of ZK53 on cell viability in H1703 (k) and SK-MES-1 (l) cells overexpressing empty vector or CLPP (n = 3 biological samples).Data are represented as mean ± SD (error bars).m Gating strategy of cell cycle analysis.The R1 was gated FSC and SSC plots of unstained cell control sample to remove cell debris, R2 was gated to exclude the doublets and cell aggregates by gating in single cells (FL2-1 vs. FL2-W).n Quantitation graph of cell cycle analysis of H520 cells upon ZK53-treatment for 48 h (n = 3 biological samples).The P values are calculated by two-sided Student's t test with confidence interval of 95%.o Apoptosis analysis of SK-MES-1 cells treated with 1 using Seahorse Analyzer (n = 4 biological samples).d-g Effect of ZK53 on the mitochondrial membrane potential in SK-MES-1 and H226 cell lines (n = 3 biological samples) (d), ATP level in H1703 cell lines (n = 3 biological samples) (e), mitochondrial ROS in SK-MES-1 and H226 cell lines (n = 3 biological samples) (f), and mtDNA copy number in H1703 and SK-MES-1 cell lines (n = 3 biological samples) (g).The P values are calculated by one-way ANOVA with Dunnett's multiple comparisons test.h Effect of ZK11, ZK53 and ADEP 4 on the thermal stability of SaClpP protein in intact cell of the S. aureus Newman strain in CETSA.The gel images shown are representative of three independent experiments.All data are represented as mean ± SD (error bars).positive.Scale bar, 50 μm.The P values are calculated by two-sided Student's t test with confidence interval of 95%.f Schematic illustration of ATM-mediate DNA damage response pathway.g Effect of ZK53 and ONC201 on Rb phosphorylation, regulatory proteins involved in the cell cycle, and DDR-related proteins in H1703 cells in immunoblot analysis.The images shown are representative of three independent experiments.h Effect of ONC201 on gene expression of E2F targets in H1703 cells measured by qPCR (n = 3 biological samples).The P values are calculated by one-way ANOVA with Dunnett's multiple comparisons test.i Effect of ZK53 and ONC201 on the reported ONC201-activated pathways in H1703 cells in immunoblot analysis.The images shown are representative of three independent experiments.j Effect of Y118A ClpP overexpression on the ZK53-related pathways in H1703 and SK-MES-1 cells in immunoblot analysis.The images shown are representative of three independent experiments.

Table 1 .
Data collection and refinement statistics.

Table 7 . Primer sequences used in this study.
N/A, not availableSupplementary Table6.Information about the bacteria strains used in this work.